Microspheres, microparticles, or beads, have become an important form of solid state substrate for detecting, analyzing and identifying a wide range of biomolecules. Advantages of using microspheres compared to traditional plate-based assay methods include their large surface area, easiness of mixing with a solution and re-collecting, availability of different chemistry to functionalize the beads and control the density and orientation of the reactive ligands on the beads. Preparation of the beads can be easily scaled up with consistent quality of beads. Different types of microspheres can be selected based on specific requirements of the assay protocol and signal detection method. Among the most widely used microspheres, there are silica beads, magnetic beads, polystyrene beads and latex beads.
In a typical bead-based assay, for example, magnetic bead-based immuno-assay, magnetic microspheres functionalized with a specific capture antibody will be first incubated with a fluid sample in which the suspect antigen may present, and then a solution of detection antibody conjugated with an enzyme, and finally solution of enzyme substrate to generate a detectable signal. During the process, in between different incubations, stringent washing of the beads are critical for accurate results. Bead washing is normally accomplished by repeated cycles of bead collection, aspiration, and re-suspension, through centrifugation, magnetic aggregation, or vacuum assisted filtration. All of these methods are lengthy, repetitive, and need extra equipment operation. After the assay, recollecting and measuring the signals of the microspheres is also a big challenge, especially for assays using small amount of beads and multiple washing steps, because loss of beads is inevitable in most cases. Similar protocols are used for many cell-based assays to examine the biomolecules on the cell membrane or inside the cells. Commonly used lab tools include multiwell filtration devices which utilize vacuum or centrifugation force to extract the liquid out of the filtration well.
Dot blot is another widely used membrane-based biomolecule analysis method. In this format, typically, one or more specific biomolecules such as antibodies, antigens, and nucleic acids, are immobilized on a porous membrane in an array of small confined area. The blotted membrane is then placed in a serial of buffer and solutions for membrane blocking, affinity binding, rinsing, and signal development. The entire process, including multiple lengthy shaking and washing steps, takes more than two hours or even overnight to finish. A commonly used apparatus for dot blot assay relies on a vacuum source to suck solution through the membrane, but the apparatus set up is complicated, expensive, and need to be assembled, disassembled and cleaned for every use. Only a limited number of membranes can be processed using such device. Cross-contamination is hard to avoid in these methods because one continuous piece of membrane is used for multiple assay dots. Therefore, there are needs in the art for apparatus and methods that can simplify the washing and shorten the total assay time for both high throughput analysis platform and rapid test for a few samples in research and clinical labs.